Covalent Proteins as Targeted Radionuclide Therapies Enhance Antitumor Effects

Molecularly targeted radionuclide therapies (TRTs) struggle with balancing efficacy and safety, as current strategies to increase tumor absorption often alter drug pharmacokinetics to prolong circulation and normal tissue irradiation. Here we report the first covalent protein TRT, which, through reacting with the target irreversibly, increases radioactive dose to the tumor without altering the drug’s pharmacokinetic profile or normal tissue biodistribution. Through genetic code expansion, we engineered a latent bioreactive amino acid into a nanobody, which binds to its target protein and forms a covalent linkage via the proximity-enabled reactivity, cross-linking the target irreversibly in vitro, on cancer cells, and on tumors in vivo. The radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining rapid systemic clearance. Furthermore, the covalent nanobody conjugated to the α-emitter actinium-225 inhibits tumor growth more effectively than the noncovalent nanobody without causing tissue toxicity. Shifting the protein-based TRT from noncovalent to covalent mode, this chemical strategy improves tumor responses to TRTs and can be readily scaled to diverse protein radiopharmaceuticals engaging broad tumor targets.


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Waters Xbridge BEH C4 column (300 Å, 3.5 μm, 2.1 mm x 50 mm). An acetonitrile gradient (30-71.4%) with 0.1% formic acid was used with a 0.2 mL/min constant flow rate at RT. Data was obtained at a rate of 1 sec/scan between 10000 to 18000 m/z. MassLynx mass spectrometry software was used to deconvolute the spectra. NbHER2 mass was calculated with the pelB leader sequence removed and the formation of two disulfide bonds.
For tandem mass spectrometry, the peptides were analyzed and sequence using a Q Exactive Orbitrap interfaced with Ultimate 3000 LC system. To prepare the peptide, 5 µg of NbHER2 (FSY) was incubated with 12.5 µg of HER2 ECD for 16 h at 37 °C. The cross-linking reaction was then subsequently digested with trypsin. The trypsin digested cross-link peptide was then injected into an Ace UltraCore super C18 reverse-phase column (300 A, 2.5 μm, 75 mm x 2.1 mm). An acetonitrile gradient (5-95%) with 0.1% formic acid was used with a 0.2 mL/min constant flow rate at RT. Stepped collision energy HCD was used to fragmentize the cross-linked peptide with a normalized collision energy of 28, 30, 35 eV. Survey scans were acquired at a resolution of 70,000 at m/z 200 on the Q Exactive. Cross-linked peptides were searched with pLink 2 and OpenUaa search engine. 3,4

Cross-linking of NbHER2 with HER2 ECD in vitro
Recombinant extra-cellular domain (ECD) of HER2 receptor was purchased from Abcam (Cat# ab168896). Purified 1 µM NbHER2 (WT) or NbHER2 (FSY) was incubated with 1 µM HER2 in 10 uL 1 X PBS, pH 7.4 for 4 h or indicated time points at 37 °C. After incubation, 4x Laemmli sample buffer (Bio Rad, Cat# 161-0747) with 2-mercaptoethanol was added into the incubation and heated at 95 °C for 10 min. The samples were separated on SDS-PAGE and either analyzed by Coomassie blue staining or Western blot. For Western blotting, the PVDF membrane was blocked with 5% milk for 1 h at RT while rocking. The membrane was then treated with 1:10000 anti-his monoclonal antibody (Proteintech #HRP66005) in 5% milk at RT while rocking. The membrane was then washed three times with PBST before imaging.

Biolayer interferometry (BLI) measurement
The association kinetics between human HER2 and NbHER2 was measured with BLI using the  or NbHER2 (FSY) and 30 mg L-lysine was intravenously injected into each mouse. The mice were sacrificed at 6 h post-injection and the NCI-N87 tumor was excised from the mice. The tumors were added 500 µL of RIPA and 1x protease inhibitor and was homogenized and lysed. The cell lysates were analyzed with Western blot as described above.

I Radiolabeling of NbHER2
NbHER2 (WT) or NbHER2 (FSY) was labeled with 124 I by using the direct iodination strategy. In a Pierce iodination tube, 3.0 mCi of Na 124 I was added and the pH was adjusted to 7 with 100 µL HEPES (1 M). Three mg of NbHER2 (WT) or NbHER2 (FSY) were added to the iodination tube and the reaction was carried out for 20 min at room temperature with frequent shaking. To check the radiolabeling efficiency, instant thin layer chromatography (iTLC) was performed using Whatman filter paper and 20 mM citric acid as mobile phase. The radiolabeled proteins were purified using G25 columns and PBS.
Buffer for 120 s, after which the sensor was incubated with varying concentrations (12.5, 25, 50, 100, and 200 nM) of NbHER2(WT) or NbHER2(FSY) (association step) for 90 s, followed with dissociation step in the Kinetic Buffer for 400 s. Data was fitted for a 1:1 stoichiometry and kinetic rate constant was calculated using the built-in software.

Preparation of Macropa-PEG4-NbHER2 and 225 Ac radiolabeling
Macropa-PEG4-TFP ester was synthesized using a previously reported method. 5  In vivo 225 Ac-NbHER2(WT) and 225 Ac-NbHER2(FSY) therapy studies NCI-N87 tumors were xenografted in mice as described above. Once the xenografted-tumor reached 100-300 mm 3 (Day 0), the mice were injected with 0.8 ± 0.2 µC 225 Ac labeled nanobodies (n = 8 for NbHER2 (FSY), n = 7 for NbHER2 (WT)) or Saline (n=5). The mice were also coinjected with 30 mg of L-lysine per mice. On Day 7, the mice were coinjected again with 0.8 ± 0.2 µC 225 Ac labeled nanobodies or saline and 30 mg of L-lysine per mice. The tumor growth was with a digital caliper in two dimensions, and tumor volume was calculated using the formula: tumor volume = length x width 2 /2. At day 26, the mice were sacrificed for analysis. The liver, kidney, heart, and bone were dissected and processed for hematoxylin and eosin (H&E) staining and analysis. The microscopic images of the H&E slides were analyzed by a trained pathologist (E.C.) and no abnormalities were observed with the tissues.
Dynamic PET acquisition was used to measure the standardized uptake value (SUV) in blood in percent injected dose per cm 3 (% ID/cc) at indicated time points. The data were fitted with two phase exponential decay to yield the t1/2 values. Error bars represent 95% CI; n = 3 mice.